Figure 5.

mTOR-independent 4E-BP1 phosphorylation in SW620 cells. (A) BEZ235 inhibits mTOR kinase activity toward 4E-BP1 in both SW480 and SW620 cells. SW480 and SW620 cells were treated with 100 nM BEZ235 or drug vehicle control (DMSO) for 6 h. mTOR was immunoprecipitation and assayed for in vitro kinase activity toward bacterial recombinant S6K1 and GST-4E-BP1 by western blot. (B) siRNA-mediated knockdown of mTORC1 inhibits 4E-BP1 phosphorylation in SW480 but not SW620 cells. SW480 and SW620 cells were transfected with siRNA targeting mTOR, raptor and rictor, respectively, for 48 h. Inhibition of mTOR signaling was analyzed by western blot.